A subset of cytochrome P450 enzymes perform the first and rate-limiting step in the clearance of foreign small molecule drugs and toxins from the human body, while others play key roles in endogenous pathways. Of necessity the former evolved the flexibility to bind and oxidize a broad range of small molecule chemical scaffolds, while the latter appear to be less flexible and have more substrate specificity. What we know about the structures of all of these membrane proteins has been determined solely by X-ray crystallography. This approach provides detailed information about atomic-level protein/ligand interactions, but has not been applied across the human spectrum of P450 enzymes and does not capture the range of conformations these enzymes are capable of adopting or their interactions with other proteins. Thus application of a cross-section of structural techniques is essential to provide the information needed to understand which P450 enzymes bind which small molecules, how they are bound, and what the products will be. This information is critical for understanding drug/toxin metabolism to forms that may be either active or inactive, adverse interactions of two drugs at the same P450 active site, and endogenous pathways related to diverse diseases. The applicant's long-term research goal is to promote understanding of the structure/function principles that control substrate and inhibitor interactions with P450 enzymes, in order that this information can be exploited to more effectively prevent and treat human disease. The objective of this proposal is to generate structures of new human cytochrome P450 enzymes with the critical components of the catalytic system: their ligands, redox partner proteins, and eventually the membrane. A number of human cytochrome P450 enzymes do not have structures available and none have structures with their catalytic partner proteins. These are gaps we propose to bridge using the following approaches, building on our previous structural expertise with more than 20 human cytochrome P450 enzymes and a collaboration with cryo-electron microscopist Dr. Melanie Ohi. Specific aim 1: Determine structures of human P450 enzymes. A structure exists for only about half of the 57 human cytochrome P450 enzymes. Many of those without structures are involved in key homeostatic pathways involving bile acids, fatty acids, eicosanoids, and vitamins, impeding our understanding of a number of corresponding diseases. We take advantage of this R37 extension opportunity to propose a small-scale structural genomics project to close the gap by determining structures of as many of these P450 enzymes as possible. As we have done successfully for many other human P450 enzymes, we will 1) engineer synthetic genes in ways that usually produce P450 holoproteins, 2) undertake expression and purification trials, and 3) subject those yielding enough active P450 protein to crystallization for X-ray structure determination. We will initially focus on those responsible for bile acid and fatty acid synthesis as we have additional expertise and tools to support success in these areas. As specific ligands can be critical for stability and crystallization, we will use a range of nonspecific luminescent P450 substrates to screen ligands that bind each P450 tightly and are likely to facilitate crystallization, as has been successful for CYP1A 1. In cases where protein is generated but crystallization does not occur (CYP2S1, CYP2W1, CYP3A7, CYP2F currently in hand), we will initiate structures using cryoEM. Preliminary studies in collaboration with Dr. Melanie Ohi have very quickly yielded a small data set for CYP3A4 with 2D resolution good enough to visualize alpha helices. Even if crystallization is successful with a particular P450 enzyme, this latter approach is independently valuable as it is likely to reveal P450 open conformations that must exist but are usually not obtained in crystals. Specific aim 2: Determine structural effects of cytochrome bs and reductase binding to human P450 enzymes. There are no structures of b5 or reductase binding to any human P450 enzymes. Over the last grant period we used NMR to identify the two b5. surfaces that differentially bind individual cytochrome P450 enzymes. The next step proposed is to use NMR to identify the opposing surfaces on individual P450 enzymes and the effects that b5 binding has on the conformation of these P450 enzymes and their interactions with ligands. We know from our previous studies that when b5. binds CYP17 A1 that major conformational changes occur on the opposite face of the P450, which is normally involved in membrane binding and ligand entry/exit from the active site. It is unclear if this is generally true. Advanced labeling techniques and substrate-directed NMR will be used to answer these questions. Finally, we will employ our P450/redox partner fusion constructs to elucidate the feasibility of determining structures of selected complexes using cryoEM. Overall, our ability to probe P450 structure using orthogonal techniques, supported by the preliminary data and expertise already developed under this grant, uniquely qualify us to expand research into these areas. Upon completion, we expect to have significantly expanded the structures available for multiple human P450 enzymes of significant interest with respect to a broad range of human diseases.